Rapid and efficient regeneration of transgenic plants

ABSTRACT

A rapid transformation regeneration system is disclosed. This system takes two-three months to obtain transgenic plants. Transformation efficiencies are very high. This system also has been demonstrated with several different selecting systems and is particularly useful for transforming wheat.

The present invention relates to genetically engineered plants. Inparticular it relates to a method for regenerating plant cells whichhave been transformed.

BACKGROUND

During the past decade, it has become possible to transfer genes from awide range of organisms to crop plants by recombinant DNA technology.This advance has provided enormous opportunities to improve plantresistance to pests, diseases and herbicides, and to modify biosyntheticprocesses to change the quality of plant products (Knutson et al., PNAS,USA 89, 2624-2628, (1992); Piorier et al., Science, 256, 520-523,(1992); Vasil et al., Bio/Technology, 10, 667-674, (1992)). However, theavailability of an efficient transformation method to introduce foreignDNA has been a substantial barrier for most monocot species, includingmaize, rice, oat, barley, and particularly wheat.

Two alternative transformation methods are currently used for monocotspecies: direct DNA transfer into isolated protoplasts andmicroprojectile-mediated DNA delivery (Shimamoto et al., Nature, 338,274-276, (1989); Fromm et al., Bio/Technology, 8, 833-839, (1990)).

The protoplast methods have been widely used in rice, where DNA isdelivered to the protoplasts through liposomes, PEG, andelectroporation. While a large number of transgenic plants have beenrecovered in several laboratories (Shimamoto et al., (1989); Datta etal., Bio/Technology, 8, 736-740, (1990)), the protoplast methods requirethe establishment of long-term embryogenic suspension cultures. Someregenerants from protoplasts are infertile and phenotypically abnormaldue to the long-term suspension culture (Davey et al., J. of Exp.Botany, 42, 1129-1169, (1991); Rhodes et al., Science, 240, 204-207,(1988)).

The microprojectile-mediated DNA delivery method may use immatureembryos or immature embryo derived calli as target tissues. Transgenicplants have been recovered from the microprojectile bombardment methodin maize, oat, barley and wheat (Gordon-Kamm et al., Plant Cell, 2,603-618, (1990); Somers et al., Bio/Technology, 10, 1589-1594, (1992);Wan et al., Plant Physiol., 104, 37-48 (1994); Vasil et al. (1992)).

The microprojectile bombardment method generally takes 10 to 15 monthsto obtain transgenic plants (Gordon-Kamm et al., (1990); Vasil et al.(1992)). Even with the more recent improvements in transformationmethods using immature embryos as target tissues, it still requires 4 to6 months to recover transgenic plants (Weeks et al., Plant Physiol.,102, 1077-1084, (1993); Vasil et al., (1992); Vasil et. al.,Bio/Technology, 11, 1153-1158 (1993); Becker et al., Plant J., 5,299-307, (1994). Moreover, these methods suffer frequently from a lossin fertility in the recovered plants (Vasil et al., (1993); Becker etal., (1994)). Furthermore, the transformation frequency by these methodsis very low, about one event from every thousand bombarded embryos. Thistransformation efficiency is too low for genetic studies and forcommercial applications.

Thus, there is a need not only for a more rapid method of regeneratingtransformed plant tissue, there is also a need for a method that retainsfertility in the resulting plants and produces a higher transformationefficiency.

SUMMARY OF THE INVENTION

The present invention provides a rapid and efficient transformation andregeneration system. The present invention is particularly useful withthe transformation and regeneration of wheat plants. Plants regeneratedfrom this system are phenotypically normal and fully fertile. Thetransgenes are transmitted to R₁ progeny in a Mendelian fashion.

In a preferred embodiment, the present invention provides a rapid andefficient regeneration system for monocot crop transformation usingproliferated immature embryos as target tissues. The new system takesless than two months to obtain transgenic plants. Transformationfrequencies by the new system are 5 to 100 times higher than the currentmethods used in other laboratories. This new system can be used with avariety of selectable marker systems, including selection usingherbicides, such as glyphosate and bialaphos, as well as antibioticssuch as kanamycin.

The present invention provides a method for regenerating a transformedmonocotyledonous plant to contain foreign DNA comprising the steps of:

a) isolating regenerable tissue from the plant;

b) inserting into the regenerable tissue the foreign DNA where theforeign DNA comprises a selectable DNA sequence, where the sequence canfunction in a regenerable tissue as a selection device;

c) between about one day to about three weeks after step b) placing theregenerable tissue from step b) in a medium capable of producing shootsfrom the tissue where the medium further contains a compound used toselect regenerable tissue containing the selectable DNA sequences; and

d) after at least one shoot has formed from step c), transferring theshoot to a second medium capable of producing roots from the shoot.

DETAILED DESCRIPTION OF THE INVENTION

The present invention can be used with any plant species. It isparticularly useful for monocot species. More particularly, it is usefulin plant species which cannot remain in a callus state for long periodsof time without losing the ability to regenerate. One particularlyuseful species in the present invention is wheat.

The present invention, when applied to wheat, has the advantage of beinggenotype independent. That is, it can be used with any type of wheatvariety, including both winter and spring wheat. It can be used toproduce transgenic wheat plants from spring cultivars, such as, forexample, Bobwhite and Marshall as well as winter cultivars, such as, forexample, Neeley.

The present invention is used to introduce foreign DNA into regenerableplant tissue. Any type of foreign DNA can be inserted into the plantspecies using the method of the present invention. Generally, "foreignDNA" can be defined to include any type of DNA which is inserted into aplant cell from outside the plant cell. Methods for inserting DNA intoplant cells are generally well known, such as a bombardment using adevice described in U.S. Pat. No. 5,179,022.

The type of DNA included in the foreign DNA can include DNA whichalready is present in the plant cell, DNA from another plant, DNA from adifferent organism, or a DNA generated externally, such as a DNAsequence containing an antisense message of a plant gene, or a DNAsequence encoding a synthetic version of a gene where the nucleotidesequence has been modified.

In one preferred embodiment, the foreign DNA contains a DNA sequencewhich can function in a regenerable plant tissue as a selection device.Such DNA can include a gene which would function in a regenerable planttissue to produce a compound which would confer upon the plant tissueresistance to an otherwise toxic compound. These genes are well known inthe art and can confer resistance to compounds such as antibiotics likekanamycin (Dekeyser et al., Plant Physiol., 90, 217-223, (1989)), andherbicides like glyphosate (Della-Cioppa et al., Bio/Technology, 5,579-584 (1987)) and bialaphos (Vasil et al., (1992)). Other selectiondevices can be used within the scope of the present invention.

The first step in the present invention is to isolate regenerable tissuefrom a plant. Any regenerable plant tissue can be used in accordancewith the present invention. Regenerable plant tissue generally refers totissue which after insertion of foreign DNA can be regenerated into adifferentiated plant. For example such tissues can include calli and/orembryoids from anthers (Zhou and Konzak, Crop Sci., 29, 817-821 (1989)),microspores (Ziauddin et al., Plant Cell Rep., 11, 489-493 (1992)),inflorescences (Barcelo et al., Plant Journal, 5, 583-592, (1994)) andleaf tissues (Conger et al., Plant Cell Reports, 6, 345-347, (1987)).

In one embodiment of the present invention, an immature embryo from aplant is used as a starting material. Immature embryos can be producedusing known method described in the art. For instance, the production ofwheat immature embryos is described by Weeks et al., (1993) and Vasil etal., (1993).

In another preferred embodiment of the present invention, theregenerable plant tissues are calli. The preferred calli are embryogeniccalli. Embryogenic calli are produced from immature embryos. These callican be produced by isolating and culturing immature embryos on anutrient media with carbohydrate and plant growth regulators. In thepreferred embodiment of the present invention, when producingembryogenic calli from wheat, the elimination of embryo axis asdescribed by Nehra et al., Plant J., 5, 285-297, (1994) is notnecessary.

Callus producing medium are well known in the art and any culture mediumor preparation method can be used. In the preferred embodiment, wherewheat calli are prepared, a wheat immature embryo is cultured for 1 dayup to one month, preferably for 4 to 7 days, on a modified MS mediumcomprising about 40 g/l realrose and about 2 mg/l 2,4-D. In anotherembodiment, the 2,4 D can be replaced by a combination of 0.5 mg/l 2,4-Dand 2.2 mg/l picloram. The medium is solidified by 2 g/l GELRITE or 4g/l low melting agarose.

Once the regenerable plant tissue is isolated, the second step of themethod is introducing the foreign DNA into the plant tissue. Thisprocess is also referred to herein as "transformation." Any method canbe used to insert the foreign DNA into the regenerable plant tissue.Such methods include bombardment (Weeks et al., (1993); Vasil et al.,(1992)), Agrobacterium transformation (Chan et al., Plant MolecularBiology, 22, 491-506, (1993)), electroporation of regenerable tissues(Shillito et. al., Bio-Technology, 3, 1099-1103, (1985)) andprotoplast-facilitated gene delivery (Shimamoto et al., (1989); Datta etal., (1990)).

In a preferred embodiment, the regenerable tissue is transformed usingthe bombardment method. In this embodiment, it is also preferred that acallus tissue, most preferably an embryogenic callus, is used. Afterbombardment, this callus can be grown for a short period of time priorto regeneration or selection or, in accordance with a preferredembodiment of the invention, can immediately be subjected to bothregeneration and selection conditions. With other transformationmethods, this period may or may not be desirable, depending upon theselection method used.

In one embodiment of the invention, the regenerable tissue is grown fora short period after bombardment. The medium used for this growth periodpreferably does not contain any selection device or any medium capableof producing shoots. The use of a growth period depends upon theselection device used. Some selection devices benefit from the use oflarger or older callus tissue before selection is applied. This growthperiod can be any period of time, but generally ranges from about 1 dayto about one month. However, in the preferred embodiment of theinvention, this growth period should be short, if one is used at all,and generally no more than about three weeks, preferably no more thanabout two weeks and most preferably no more than about one week andspecifically from about 1 to about 7 days after bombardment. A growthperiod is not required in the present invention.

In another embodiment of the invention, the regenerable plant tissue canbe subjected during this period after bombardment to a short period ofselection prior to exposure of the regenerable tissue to a mediumcapable of producing shoots. Any selection compounds can be used duringthis period consistent with the selectable DNA sequence inserted intothe regenerable tissue. Such compounds include paromomycin, glyphosateand bialaphos.

While this selection period prior to regeneration is not required underthe claimed invention, if used, this period can range from about 1 dayto about two weeks. More preferably, this period will range from about 1to 7 days.

After transformation, the regenerable plant tissue is placed in a mediumcapable of producing shoots from the regenerable tissue where the mediumfurther contains a compound used to select regenerable tissue containingthe selectable DNA sequences. This is in contrast to the prior art whereregenerable plant tissue is generally subjected first to an extendedperiod of selection prior to exposure of the regenerable tissue to amedium capable of producing shoots.

The medium used in this step can be any medium which permits theformation of shoots from the regenerable tissue. In one embodiment, ashoot-producing compound is added to the medium. These shoot-producingcompounds are well known in the art (Murashige and Skoog, Physiol.Plant, 15, 473-497, (1962); Kasha et al., Gene Manipulation in PlantImprovement II, 213-239, (1990)). Such compounds include weak plantgrowth regulators and include IAA, IBA, and BA at low concentrations(Becker et al., (1994); Vasil et al., (1992)). In another embodiment ofthe invention, a medium free of a plant growth regulator can be used toinduce shoot formation (Weeks et al., (1993)).

In a preferred embodiment, where an embryogenic wheat calli is to beregenerated, the medium comprises a modified MS medium with 0.2 mg/l2,4-D (Murashige and Skoog, (1962); Wan and Lemaux, (1994)).

The regenerable plant tissue is generally placed in this medium asquickly as possible in the present invention after transformation.Generally, this can range from about 1 day to about three weeks, butpreferably from about 1 day to about two weeks. Most preferably thetissue is transferred to this medium from about one week to about twoweeks after transformation. In most instances, the transfer will occurbetween about 5 and about 11 days.

The compound used to select regenerable tissue containing the selectableDNA sequences can be any of a variety of well known selection compounds,such as antibiotics and herbicides. Preferred compounds can includekanamycin (Dekeyser et al., (1989)), glyphosate (Della-Coppa et al.,(1987)) and bialaphos (Vasil et al., (1992); Weeks et al., (1993)).

The availability of alternative selection agents is an importantrequirement for commercial application of agriculture biotechnology. Theuse of kanamycin has been less successful for cereal crops because ofthe high endogenous level of tolerance (Dekeyser et al., (1989)).Bialaphos has been widely used as a selection agent in cereal croptransformation (Weeks et al., (1993); Vasil et al., (1993); Becker etal., (1994); Nehra et al., (1994); Wan and Lemaux (1993)). However, itcould potentially be a disaster to exclusively use genes encodingbialaphos resistance as a selectable marker in all transformationexperiments. Other selectable markers are needed and our resultsdemonstrate that the herein described rapid regeneration system workswell with different selection agents.

After shoots have formed the shoots are transferred to a second mediumcapable of producing roots from said shoots. This medium can furthercontain a compound used to select regenerable tissue containing theselectable DNA sequences. Transfer to this medium occurs when sufficientshoots have developed, as generally known in the art. This occurs, forwheat, within 25 to 40 days after transformation.

The medium capable of producing roots can be any root-producing medium.These mediums are well known in the art (Weeks et al., (1993); Vasil etal., (1992)). One preferred root-producing medium is a modified MSmedium without any plant growth regulator (Murashige and Skoog, (1962);Zhou et al., Plant Cell Tissue and Organ Culture, 30, 78-83, (1992)).

Once roots have been formed, the plants can then be transferred to soiland grown following methods known in the art to produce seeds.

One advantage of the above described transformation and regenerationmethod is that plants obtained from this process are generally fertile.The loss of fertility among transgenic plants using prior art methods isbelieved to be attributed to the long-term cultures before and after thetransformation treatments rather than the act of transformation per se.

Another advantage of the present invention it that the currentbombardment methods require 4 to 6 months to obtain transgenic plants(Becker et al., (1994); Vasil et al., (1992), (1993); Weeks et al.,(1993)). The bombarded regenerable tissues of these prior art methodswere subcultured on selection media for 2 to 3 months or longer to allowcallus proliferation. By reducing the time of the callus proliferationculture, the rapid regeneration method described herein requires lessthan 2 months to obtain transgenic plants.

Use of the present method also caused transformed tissues to regeneratemuch faster. The regenerants were also more vigorous and healthier bothin culture and in soil.

The rapid regeneration system described herein also usually producesuniform, non-chimeric transformants. With the rapid regeneration method,embryogenic callus sectors are usually small at the stage ofregeneration. Therefore, only a single shoot is regenerated from eachcallus sector. Histochemical analysis for stable GUS activity showedthat leaf segments from different parts of the transgenic plants weregenerally uniform in GUS expression. Progeny analysis also indicatesthat most of the transgenic plants segregated at 3:1 ratios betweentolerant and sensitive plants as a single dominant gene.

The following examples describe specific embodiments of the invention.Media used are described in Table 9. The examples are provided to betterelucidate the practice of the present invention and should not beinterpreted in any way to limit the scope of the present invention.Those skilled in the art will recognize that various modifications,additions, etc. can be made to the methods described herein while notdeparting from the spirit and scope of the present invention.

EXAMPLE 1: TRANSFORMATION USING CP4 AND GOX AS SELECTABLE MARKERS

1. Immature embryo culture A spring wheat Triticum aestivum cv. Bobwhitewas used throughout this study. Stock plants were grown in anenvironmentally controlled growth chamber with 16-h photoperiod at 800μmol m⁻² s⁻¹ provided by high-intensity discharge (HID) Sylvania lights(GTE Products Corp., Manchester, N.H. 03103). The day/night temperatureswere 18°/16° C. Immature caryopses were collected from the plants 14-dafter anthesis. Immature embryos ("IE") were dissected and cultured on amodified MS medium (Murashige and Skoog salts, Gibco BRL) supplementedwith 40 g/l maltose, 0.5 mg/l 2,4-D, and 2.2 mg/l picloram (CM4). Theimmature embryos were cultured at 26° C. in the dark.

2. DNA delivery Five days after the initiation of culture, immatureembryos were transferred to an osmoticum treatment medium 4-h prior tobombardment. The osmoticum medium was the same CM4 with 0.35M mannitol.Thirty to 40 embryos were placed in the center of each plate, and theembryos were bombarded with a mixture of pMON19305 and pMON19328 at an1:1 ratio. pMON19305 contains the uidA gene whereas pMON19328 carriesthe glyphosate-tolerant CP4 and GOX genes. CP4 is a bacterial5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene which expressesan enzyme highly resistant to glyphosate. The glyphosate oxidoreductase(GOX) is a bacterial gene which degrades glyphosate into aminomethylphosphortic acid. All genes were driven by the maize ubiquitin Ubilpromoter (Christensen et al., (1992)). Each plate was bombarded twicewith a PDS 1000 powder gun. High levels of transient GUS expression wereobserved for every bombardment in every experiment, indicating that theDNA delivery method was very efficient.

3. Regeneration of glyphosate-tolerant plants After a 16-hpost-bombardment treatment on the 0.35M mannitol medium, the bombardedembryos were transferred to the CM4 medium (Table 1) for a 1-week delayof selection. At this stage, two embryos from each bombarded plate weresampled for transient GUS assays. After a one or two-week delay, theembryos were transferred to an CM4 medium containing 4 mM glyphosate.After one to two weeks of callus proliferation culture on this selectionmedium, the embryos were transferred to regeneration media containing0.1 mM glyphosate (Protocol 1). In some cases, the bombarded embryoswere directly transferred to the regeneration medium one to two weeksafter bombardment (Protocol 2). Shoots obtained from the regenerationmedia were transferred to a rooting medium containing 0.02 mMglyphosate. Tolerant plants were transferred to soil and grown in anenvironmentally controlled growth chamber as described. Two weeks later,the plants were sprayed with 8 oz/a ROUNDUP® (active ingredientglyphosate, Monsanto).

    ______________________________________                                        Process                 Timeframe                                             ______________________________________                                        Protocol 1: The rapid transformation regeneration method for                  glyphosate selection - reduced callus proliferation culture.                  Culture IE on CM4 medium                                                                               0-d                                                     ↓                                                                   Bombard embryo callus    5-d                                                     ↓                                                                   Transfer callus to CM4 + 4 mM Gt                                                                      12-d                                                     ↓                                                                   Regenerate on MMS.2 + .1 mM Gt after                                                                  19-d                                                  1 wk callus proliferation culture                                                ↓                                                                   Root on MMSO + .02 mM Gt                                                                              40-d                                                     ↓                                                                   Transfer plants to soil 60-d                                                  Protocol 2: The rapid transformation regeneration method for                  glyphosate selection - eliminated callus proliferation culture.               Culture IE on CM4 medium                                                                               0-d                                                     ↓                                                                   Bombard embryo callus    5-d                                                     ↓                                                                   Regenerate on MMS.2 + .1 mM Gt                                                                        12-d                                                  after 1 wk delay                                                                 ↓                                                                   Root on MMSO + .02 mM Gt                                                                              35-d                                                     ↓                                                                   Transfer plants to soil 60-d                                                  ______________________________________                                    

On callus proliferation media, there was no visual difference onembryogenesis between the tissues bombarded with and without plasmidDNA. However, when transferred to regeneration medium, green shoots wereregenerated from the transformed embryos, whereas no shoots wererecovered from controls bombarded without plasmid DNA. We were able torecover glyphosate tolerant shoots from most experiments. On average,about 1-2% bombarded embryos produced glyphosate-tolerant plants (seeTable 1). Under our experimental conditions, transformation efficiencywith the old method was very low. About 0.05% bombarded embryos producedglyphosate tolerant plants (Table 2). Transformation efficiency from therapid method was 25-fold higher than that from the old method. Thetransformation frequency from the old method was equivalent to those ofbialaphos selection previously reported by other groups (Vasil et al.1992, 1993; Weeks et al. 1993; Nehra et al. 1994).

                  TABLE 1                                                         ______________________________________                                        Glyphosate-tolerant plants recovered from different selection                 regeneration regimes by the rapid regeneration method.                                       Expt/                  #GUS                                    Treatment      Trt#    #I.E.   #events                                                                              pos.                                    ______________________________________                                        1 wk delay, direct regen.                                                                    37-2    300     3      2                                                      42-2    350     3      3                                                      43-2    400     5      5                                       2 wk delay, direct regen.                                                                    37-3    300     1      1                                                      42-3    350     3      2                                                      43-3    400     7      3                                       1 wk delay/1 wk callusing                                                                    43-4    400     11     11                                      1 wk delay/2 wk callusing                                                                    42-4    175     3      3                                       Total                  2675    36 (1.3%)                                      ______________________________________                                         Expt: experiment; Trt: treatment; I.E.: immature embryos; GUS:                glucuronidase.                                                           

                  TABLE 2                                                         ______________________________________                                        A summary of glyphosate tolerant plants produced from the old                 and the rapid regeneration methods.                                           Methods                                                                              #Expt    #I.E.    #events Freq.  Months                                ______________________________________                                        Old    14       13,000   6       0.05%  4.5                                   Rapid  3         2,675   36      1.3%   2.0                                   ______________________________________                                         Expt: experiment; Trt: treatment; I.E.: immature embryos; Freq.:              frequency.                                                               

4. Enzyme assay of CP4 and GOX Transformed Plants Crude proteins wereextracted from fresh leaves of transgenic plants following a BioRadmethod. CP4 and GOX proteins were probed by antibodies, and calculatedas percentages of total proteins. The transgenic plants contained0.007-0.160% and 0.004-0.062% GOX, which were equivalent to a previouslyconfirmed transgenic plant (Table 3). Five transgenic plants had no CP4expression. Glyphosate tolerance of these plants was probably conferredby the GOX gene.

                  TABLE 3                                                         ______________________________________                                        Stable GUS expression and percent CP4 and GOX protein                         contents of glyphosate tolerant plants.                                       Transgenic lines                                                                          Stable GUS   % CP4   % GOX                                        ______________________________________                                        42-2-02     +            0.079   0.014                                        42-3-01     +            0.017   0.009                                        42-3-02     -            0.009   0.051                                        42-4-01     +            0.160   0.017                                        42-4-02     +            --      0.007                                        42-4-03     +            0.106   0.022                                        43-2-01     +            0.079   0.038                                        43-2-02     +            0.037   0.017                                        43-2-03     +            0.017   0.018                                        43-2-05     +            0.028   0.028                                        43-3-01     +            0.030   0.017                                        43-3-02     -            0.007   0.004                                        43-3-03     -            0.008   0.004                                        43-3-04     +            --      0.011                                        43-3-05     -            --      0.020                                        43-3-06     +            0.007   0.004                                        43-3-07     -            --      0.045                                        43-3-08     -            --      0.027                                        43-4-01     +            0.070   0.062                                        43-4-02     +            0.022   0.012                                        43-4-03     +            0.019   0.042                                        43-4-04     +            0.151   0.035                                        43-4-05     +            0.034   0.044                                        43-4-06     +            0.076   0.028                                        43-4-11     +            0.016   0.017                                        Bobwhite                 0.001   0.001                                        16-5(CK+)                0.028   0.011                                        ______________________________________                                    

5. Progeny analysis of glyphosate-tolerant plants Immature embryos fromthe glyphosate-tolerant plants were isolated 20-d after anthesis andcultured on a germination medium (MMS medium without plant growthregulator) with 0.02 mM glyphosate. Germinated and non-germinatedembryos were separated and recorded 10 day after the culture and thedata were analyzed by X² test for 3:1 segregation (Table 4). X² testindicated that the transgene segregated at a 3:1 ratio as expected. Thetolerant plants were then transplanted to soil and sprayed with 8 oz/aROUNDUP®. Individuals germinated on the selection media were alsotolerant to the spray.

                  TABLE 4                                                         ______________________________________                                        Germination test of embryos from glyphosate tolerant R.sub.0 plants.          Transgenic                                                                            % CP4   % GOX    Germination test                                     lines   protein protein  Tolerant                                                                             Sensitive                                                                            3.1 prob.                              ______________________________________                                        93-42-2-2                                                                             0.079   0.014    31     11     >0.9                                   93-43-2-1                                                                             0.079   0.038    27     26     <0.01                                  93-43-2-3                                                                             0.017   0.018    48     9      >0.1                                   93-43-2-5                                                                             0.028   0.028    33     17     >0.1                                   93-43-4-1                                                                             0.070   0.062    24     10     >0.1                                   93-43-4-2                                                                             0.022   0.012    6      32     <0.01                                  93-43-4-3                                                                             0.019   0.042    39     13     >0.9                                   93-43-4-4                                                                             0.151   0.035    9      29     <0.01                                  93-43-4-5                                                                             0.034   0.044    52     6      <0.01                                  Bobwhite                 0      45                                            (control)                                                                     ______________________________________                                    

EXAMPLE 2: Transformation Using the bar Gene As a Selectable Marker

1. Transformation and selection Transformation method for the bar genewas essentially the same as the CP4 and GOX genes. Immature embryos werebombarded with the pAHC25, which carries the bar and uidA genes. Bothgenes were driven by the Ubiq1 promoter. The bar gene encodesphosphinothricin acetyltransferase (PAT) that acetylatesphosphinothricin, the active ingredient of the non-selective herbicideBasta® (Hoechst AG). The bombarded embryos were transferred to the MMS2medium with 4 mg/l bialaphos one day after the bombardment (Protocol 3).

    ______________________________________                                        Protocol 3: The rapid regeneration system for bar gene                        transformation.                                                               Process                  Timeframe                                            ______________________________________                                        Culture IE on MMS2 medium                                                                               0-d                                                    ↓                                                                   Bombard embryo callus     5-d                                                    ↓                                                                   Transfer to MMS2 + 4 mg/l bialaphos                                                                     6-d                                                    ↓                                                                   Transfer to MMSO + 4 mg/l bialaphos                                                                    13-d                                                    ↓                                                                   Transfer to sundae cup on the same medium                                                              30-d                                                    ↓                                                                   Transfer plants to soil  50-d                                                 ______________________________________                                    

2. Regeneration of bialaphos-tolerant plants Following a one to two weekcallus proliferation on the MMS2 medium, the embryos were transferred toan MMS0 regeneration medium with 4 mg/l bialaphos. Shoots and plantsfrom the regeneration medium were transferred to sundae cups with thesame medium for rooting. Bialaphos tolerant plants were recovered andtransferred to soil within two months. From a total of 828 bombardedembryos in the three experiments, 566 embryos producedbialaphos-tolerant plants (Table 5). About 15% of the embryos producedsingle plant, 20% of them with 2 plants, 40% with 3 plants, and 25% with4 or more plants. Each embryo was counted as a single transformationevent regardless the number of plants recovered. One third (190 out of566) of the transformation events were GUS positive. GUS activity oftenvaried among individual events, from completely dark blue to bluestripes in vascular tissues or blue dots randomly scattered on theleaves.

Transformation frequency with bialaphos selection was much higher thanglyphosate under our experimental conditions, and 10 to 100-fold higherthan those previously reported for wheat and barley (Vasil et al. 1992,1993; Weeks et al. 1993; Nehra et al. 1994; Wan and Lemaux 1993).

                  TABLE 5                                                         ______________________________________                                        Bialaphos-tolerant plants recovered by the rapid regeneration                 system.                                                                                                  Bialaphos                                                  Time of   No. of   tolerant GUS positive                              Expt-Trt#*                                                                            callusing embryo   No.  %     No.  %                                  ______________________________________                                        Expt 44-3                                                                             2 wks     269      212  78.8  63   29.7                               Expt 46-3                                                                             1 wk      192      146  76.0  52   35.6                               Expt 46-4                                                                             2 wks     187      119  63.6  42   35.3                               Expt 48-3                                                                             1 wk      90       59   65.6  22   37.3                               Expt 48-4                                                                             2 wks     90       43   47.8  17   39.5                               ______________________________________                                         Expt: experiment; Trt: treatment; GUS: glucuronidase.                    

3. Basta® spray A sample of 20 bialaphos-tolerant plants weretransferred to soil and sprayed with 1% Basta® (200 g/l glufosinate,Hoechst AG). Control plants showed necrosis and browning 3-d after thespraying. The damaged leaves turned yellow and dried later on. Ten ofthe 20 bialaphos tolerant plants did not show any necrotic lessionsafter the spraying.

4. PAT assay The Basta® tolerant plants were analyzed for PAT activityfollowing the method of De Block et al. (EMBO J, 6, 2513-2518, 1987).All Basta® tolerant plants were PAT positive, whereas no PAT activitywas observed in Bobwhite control plants (Table 6).

5. Germination test and progeny analysis The Basta® tolerant plants grewnormally and set seeds. Immature embryos from the plants were culturedon a germination medium with 2 mg/l bialaphos. Immature embryos fromBobwhite control plants could not germinate on the bialaphos selectionmedium, whereas embryos from the Basta® tolerant plants segregated intotolerant and sensitive ones. Two of the ten plants showed 3:1segregation ratios as expected. Five had less than 3:1 segregationwhereas the other three did not produce any tolerant embryos (Table 6).It is unknown at this stage what has caused the unexpected segregation.It could be due to the small size of samples or due to gene silence.Nevertheless, the production of bialaphos tolerant plants demonstratedthat the rapid regeneration system is independent of selectable markersor any gene of interest.

                  TABLE 6                                                         ______________________________________                                        Germination test for bialaphos tolerant R.sub.0 plants recovered              from the rapid regeneration system.                                                          Germination test                                               Transgenics                                                                             GUS     PAT    Tolerant                                                                              Sensitive                                                                            3:1 prob.                             ______________________________________                                        44-3-01   -       +      0       30     na                                    44-3-02   -       +      28      8       .5-.75                               44-3-03   +       +      21      15     <.05                                  44-3-06   +       +      18      8      .25-.50                               44-3-07   -       +      14      15     <.01                                  46-3-01   +       +      0       40     na                                    46-3-02   +       +      11      31     <.01                                  46-3-05   +       +      0       48     na                                    46-3-06   +       +      4       32     <.01                                  46-3-07   -       +      14      27     <.01                                  Bobwhite (CK)                                                                           -       -      0       40     na                                    ______________________________________                                         GUS: glucuronidase; PAT: phosphinothricin acetyltransferase.             

EXAMPLE 3: Transformation Using the nptII gene As A Selectable Marker

The rapid transformation regeneration system also was demonstrated withparomomycin selection for the nptII gene.

1. Immature embryo culture A spring wheat Triticum aestivum cv. Bobwhitewas used throughout this study. Stock plants were grown in anenvironmentally controlled growth chamber with 16-h photo period at 800μmol m⁻² s⁻¹ provided by high-intensity discharge (HID) Sylvania (GTECorp.). The day/night temperatures were 18°/16° C. Immature caryopseswere collected from the plants 13 or 14-d after anthesis and cultured ona modified MS medium (Murashige and Skoog salts, Gibco BRL) supplementedwith 40 g/l maltose, 0.5 mg/l 2,4-D, and 2.2 mg/l picloram (CM4). Theimmature embryos were cultured at 26° C. in the dark.

2. DNA delivery Five days after the initiation of culture, immatureembryos were transferred to an osmoticurn treatment medium 4-h prior tobombardment. The osmoticum medium was the same CM4 with 0.35M mannitolor 0.125M mannitol and 0.125M raffinose. Approximately 40 embryos wereplaced in the center of each plate, and the embryos were bombarded witha mixture of pMON19476 and pMON19468 at an 1:1 ratio. pMON19476 containsthe enhanced 35S promoter from CaMV (Odell et al, 1985, Kay et al.1987), the NPTII gene (Fraley et al 1983), and the NOS terminator fromthe nopaline synthase gene (Fraley et al. 1983). pMON19468 carries theuidA (which encodes Beta-glucuronidase (GUS) from Escherichia coli(Jefferson et al 1986) and the NOS terminator. Both the nptII and GUSgene were driven by the 35S promoter (Odell et al, 1985, Kay et al.1987). Each plate was bombarded twice with a PDS 1000 powder gun asdescribed in detail by Klein et al 1987. High levels of transient GUSexpression (an average of 84 spots per embryo) were observed, indicatingthat the DNA delivery method was very efficient.

3. Regeneration of paromomycin-tolerant plants After a 18-hpost-bombardment treatment on the 0.35M mannitol medium or a combinationof 0.125M mannitol and 0.125M raffinose medium, the bombarded embryoswere transferred to the CM4 medium (Table 9) for a 6 or 7 day delay ofselection. At this stage, two embryos from each bombarded plate weresampled for transient GUS assays. After a 6 or 7 day delay, the embryoswere transferred to a CM4 medium containing 100, 200, or 300 mg/lparomomycin for callus proliferation. Another set of embryos weretransferred directly to regeneration medium containing 100 or 200 mg/lparomomycin (Protocol 4). In some cases, the bombarded embryos weredirectly transferred to the regeneration medium one to two weeks afterbombardment (Protocol 5). Shoots obtained from the regeneration mediawere transferred to a rooting medium containing no selective agent(Table 9). Plants were scored for GUS by histochemical analysis.Positive plants and some negative plants (controls) were transferred tosoil and grown in an environmentally controlled growth chamber asdescribed.

4. Progeny analysis of paromomycin-tolerant plants. Immature embryosfrom the paromomycin-tolerant plants were isolated 20-d after anthesisand cultured on a germination medium (MMS0 medium without plant growthregulator) with 100 mg/l paromomycin. Germinated and non-germinatedembryos were separated and recorded 10 day after the culture and thedata was analyzed by X² test for 3:1 segregation (Table 8). X² testindicated that the transgene did not segregate at a 3:1 ratio asexpected.

    ______________________________________                                        Process                 Timeframe                                             ______________________________________                                        Protocol 4: The rapid regeneration system for paromomycin                     selection - reduced callus proliferation culture.                             Culture IE on CM4 medium                                                                               0-d                                                     ↓                                                                   Bombard embryo callus    4-d                                                     ↓                                                                   Transfer callus to CM4 +                                                                               9-d                                                  100 mg/l paromomycin                                                             ↓                                                                   Regenerate on MMS ZR/NAA medium after                                                                 16-d                                                  1,2, or 3 wks callus proliferation culture                                       ↓                                                                   Root on MMSO            37-d                                                     ↓                                                                   Transfer plants to soil 72-d                                                  Protocol 5: The rapid regeneration system for paromomycin                     selection - eliminated callus proliferation culture.                          Culture IE on CM4 medium                                                                               0-d                                                     ↓                                                                   Bombard embryo callus    4-d                                                     ↓                                                                   Regenerate on MMSZR/IAA medium                                                                         9-d                                                  after 5 day delay                                                                ↓                                                                   Root on MMSO            37-d                                                     ↓                                                                   Transfer plants to soil 72-d                                                  ______________________________________                                    

On callus proliferation media, some of the callus turned white andstopped proliferating while some parts of the callus remained yellow andproliferated. Both the yellow and white callus tissues appeared compactand embryogenic. The tissues bombarded without plasmid DNA did notproliferate and bleached. When transferred to regeneration medium, greenshoots were regenerated from the transformed embryos, whereas no shootswere recovered from controls bombarded without plasmid DNA. We were ableto recover paromomycin tolerant shoots from all experiments. On average,about 0.3-4% bombarded embryos produced GUS positiveparomomycin-tolerant plants (Table 7). The protocol 4 produced 2 and 4%GUS positive plants while the protocol 5 produced 0.3 to 1% GUS positiveplants. Both protocols produced higher transformation frequencies thanthose of bialaphos selection previously reported by other groups (Vasilet al. 1992, 1993; Weeks et al. 1993).

                  TABLE 7                                                         ______________________________________                                        Paromomycin selected plants recovered from different selection                regeneration regimes by the rapid regeneration method.                        Treatment                                                                     Delay  Time     Paro                                                          Before on       Conc                                                          Selection                                                                            Callusing                                                                              mg/l   Expt/Trt#                                                                             #IE  #Plants                                                                              #gus+                              ______________________________________                                        7d     3 week   100    88-02   400  30     4                                  7d     3 week   200    88-03   400  37     3                                  7d     3 week   300    88-04   369  35     2                                  5d     0        100    91-01   162  52     6                                  5d     0        200    91-02   161  38     3                                  5d     1 week   100    91-05   365  29     1                                  5d     2 week   100    91-07   364  23     4                                  5d     3 week   100    91-09   364  21     4                                  Total                          2585 265    27                                 ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Paromomycin germination assay on R.sub.1 progeny from plants                  transformed with the nptII gene.                                              Transgenic                                                                             Scorable   Germination assay                                         lines*   Marker     #Tolerant #Sensitive                                                                            3:1 prob                                ______________________________________                                        88-04-01-01                                                                            Gus(+)     30        50      <0.01                                   88-04-02-01                                                                            Gus(-)     26        54      <0.01                                   88-24-06-02                                                                            Gus(+)     36        44      <0.01                                   88-14-04-02                                                                            Anthro(+)  37        43      <0.01                                   88-35-01-01                                                                            Anthro(+)  18        14      <0.01                                   Control BW                                                                             none       0         80      NA                                      ______________________________________                                         *Plants transformed with either:                                              pMON19476 (E35S/HSP70/NPTII) + pMON19468 (E35S/HSP70/GUS) or pMON19476        (E35S/HSP70/NPTII) + BC17 anthocyanin)                                   

                  TABLE 9                                                         ______________________________________                                        Tissue Culture Media used for wheat callus development, and                   regeneration of plant cells.                                                                                    Additional                                  Medium           Carbohydrate     Components                                  Name      MS*    per liter   pH   mg/l                                        ______________________________________                                        CM4       +      40 g maltose                                                                              5.8  500 glutamine                                                                 750 MgCl                                                                      100 casein                                                                    hydrolysate                                                                   0.5 2,4-D                                                                     2.2 picloram                                MMSZR/IAA +      40 g maltose                                                                              5.8  500 glutamine                                                                 750 MgCl                                                                      100 casein                                                                    hydrolysate                                                                   5 Zeatin riboside                                                             1 IAA                                       MMS2      +      40 g maltose                                                                              5.8  2 2,4-D                                     MMS.2     +      40 g maltose                                                                              5.8  0.2 2,4-D                                   MMSO      +      20 g sucrose or                                                                           5.8                                                               40 g maltose                                                 ______________________________________                                         *Basal MS medium described in (Zhou et al. 1993)                              2 g/l gelrite used for all media except paromomycin selection medium whic     contains 4 g/l agarose.                                                  

The above examples demonstrate the transformation method using threeselection devices. One skilled in the art would recognize that thismethod can be applied to many selection systems and that the inventionis not limited to these examples but only limited by the attachedclaims.

We claim:
 1. A method for regenerating a fertile transformed wheat plantto contain foreign DNA comprising the steps of:(a) producing regenerabletissue from said plant to be transformed: (b) transforming saidregenerable tissue with said foreign DNA where said foreign DNAcomprises a selectable DNA sequence, where said sequence functions in aregenerable tissue as a selection device; (c) between about one day toabout 21 days after step b) placing said regenerable tissue from step b)in a medium capable of producing shoots from said tissue wherein saidmedium further contains a compound used to select regenerable tissuecontaining said selectable DNA sequence to allow identification orselection of the transformed regenerated tissue; (e) after at least oneshoot has formed from the selected tissue of step c), transferring saidshoot to a second medium capable of producing roots from said shoot toproduce a plantlet; and, (e) growing said plantlet into a fertile,transgenic wheat plant wherein the foreign DNA is transmitted to progenyplants in mendelian fashion.
 2. The method of claim 1 where saidregenerable tissue is moved to the medium of step c) between from aboutone day to about two weeks after step b).
 3. The method of claim 1 wheresaid regenerable tissue is moved to the medium of step c) between fromabout five days to about eleven days after step b).
 4. The method ofclaim 1 where said selectable DNA sequence expresses an enzyme whichwill confer resistance to at least one of the group consisting ofkanamycin and paromomycin to said plant cell.
 5. The method of claim 1where said selectable DNA sequence expresses an enzyme which will conferresistance to glyphosate to said plant cell.
 6. The method of claim 1where said selectable DNA sequence expresses an enzyme which will conferresistance to bialaphos to said plant cell.
 7. A method for regeneratinga fertile transformed wheat plant to contain foreign DNA comprising thesteps of:(a) producing embryogenic calli from said plant to betransformed; (b) transforming said calli with said foreign DNA wheresaid foreign DNA comprises a selectable DNA sequence, where saidsequence functions in a regenerable tissue as a selection device; (c)between about one day to about 21 days after step b) placing saidregenerable tissue from step b) in a medium capable of producing shootsfrom said tissue where said medium further contains a compound used toselect the transformed regenerable tissue containing said selectable DNAsequences to allow identification or selection of the transformedregenerated tissue; (d) after at least one shoot has formed from theselected tissue of step c), transferring said shoot to a second mediumcapable of producing roots from said shoot to produce a plantlet; and,(e) growing said plantlet into a fertile transgenic wheat plant whereinthe foreign DNA is transmitted to progeny plants in mendelian fashion.8. The method of claim 7 where said regenerable tissue is moved to themedium of step c) between from about one day to about two weeks afterstep b).
 9. The method of claim 7 where said regenerable tissue is movedto the medium of step c) between from about five days to about elevendays after step b).
 10. The method of claim 7 where said selectable DNAsequence expresses an enzyme which will confer resistance to at leastone of the group consisting of kanamycin and paromomycin to said plantcell.
 11. The method of claim 7 where said selectable DNA sequenceexpresses an enzyme which will confer resistance to glyphosate to saidplant cell.
 12. The method of claim 7 where said selectable DNA sequenceexpresses an enzyme which will confer resistance to bialaphos to saidplant cell.